Carrière F, Withers-Martinez C, van Tilbeurgh H, Roussel A, Cambillau C, Verger R
Laboratoire de Lipolyse Enzymatique, CNRS-IFR1 UPR 9025, 31 chemin Joseph Aiguier, 13402 Marseille cedex 20, France.
Biochim Biophys Acta. 1998 Nov 10;1376(3):417-32. doi: 10.1016/s0304-4157(98)00016-1.
The classical human pancreatic lipase (HPL), the guinea pig pancreatic lipase-related protein 2 (GPLRP2) and the phospholipase A1 from hornet venom (DolmI PLA1) illustrate three interesting steps in the molecular evolution of the pancreatic lipase gene family towards different substrate selectivities. Based on the known 3D structures of HPL and a GPLRP2 chimera, as well as the modeling of DolmI PLA1, we review here the structural features and the kinetic properties of these three enzymes for a better understanding of their structure-function relationships. HPL displays significant activity only on triglycerides, whereas GPLRP2 displays high phospholipase and galactolipase activities, together with a comparable lipase activity. GPLRP2 shows high structural homology with HPL with the exception of the lid domain which is made of five amino acid residues (mini-lid) instead of 23 in HPL. The lid domain deletion in GPLRP2 allows the free access to the active site and reduces the steric hindrance towards large substrates, such as galactolipids. The role of the lid domain in substrate selectivity has been investigated by site-directed mutagenesis and the substitution of HPL and GPLRP2 lid domains. The addition of a large-size lid domain in GPLRP2 increases the substrate selectivity for triglycerides by depressing the phospholipase activity. The phospholipase activity is, however, not induced in the case of the HPL mutant with GPLRP2 mini-lid. Therefore, the presence of a full-length lid domain is not the unique structural feature explaining the absence of phospholipase activity in HPL. The 3D structure of the GPLRP2 chimera and the model of DolmI PLA1 reveal a higher hydrophilic/lipophilic balance (HLB) of the surface loops (beta5 loop, beta9 loop, lid domain) surrounding the active site, as compared to the homologous loops in HPL. This observation provides a potential explanation for the ability of GPLRP2 and DolmI PLA1 to hydrolyze polar lipids, such as phospholipids. In conclusion, the beta5 loop, the beta9 loop, and the lid domain play an essential role in substrate selectivity towards triglycerides, phospholipids and galactolipids.
经典的人胰脂肪酶(HPL)、豚鼠胰脂肪酶相关蛋白2(GPLRP2)和黄蜂毒液中的磷脂酶A1(DolmI PLA1)展示了胰脂肪酶基因家族在分子进化过程中朝着不同底物选择性发展的三个有趣阶段。基于HPL和GPLRP2嵌合体的已知三维结构,以及DolmI PLA1的建模,我们在此回顾这三种酶的结构特征和动力学特性,以便更好地理解它们的结构 - 功能关系。HPL仅对甘油三酯具有显著活性,而GPLRP2则具有高磷脂酶和半乳糖脂酶活性,同时具有相当的脂肪酶活性。GPLRP2与HPL具有高度的结构同源性,不同之处在于其盖子结构域由五个氨基酸残基组成(迷你盖子),而HPL的盖子结构域由23个氨基酸残基组成。GPLRP2中盖子结构域的缺失使得活性位点能够自由暴露,并减少了对大尺寸底物(如半乳糖脂)的空间位阻。通过定点诱变以及HPL和GPLRP2盖子结构域的替换,研究了盖子结构域在底物选择性中的作用。在GPLRP2中添加大尺寸的盖子结构域通过抑制磷脂酶活性增加了对甘油三酯的底物选择性。然而,在具有GPLRP2迷你盖子的HPL突变体中并未诱导出磷脂酶活性。因此,全长盖子结构域的存在并非解释HPL中缺乏磷脂酶活性的唯一结构特征。与HPL中的同源环相比,GPLRP2嵌合体的三维结构和DolmI PLA1的模型显示,围绕活性位点的表面环(β5环、β9环、盖子结构域)具有更高的亲水/亲脂平衡(HLB)。这一观察结果为GPLRP2和DolmI PLA1水解极性脂质(如磷脂)的能力提供了一个潜在的解释。总之,β5环、β9环和盖子结构域在对甘油三酯、磷脂和半乳糖脂的底物选择性中起着至关重要的作用。